Protein kinases are key regulators in many cellular process like signal transduction, proliferation, cell cycle regulation, differentiation and survival/apoptosis as well as pathophysiological alterations within these processes causing diseases. Thus, protein kinases constitute an important target class for therapeutic intervention (P. Cohen, Nature Rev Drug Discovery 1, 309, 2002; T. G. Cross, et al., Exp. Cell Res. 256, 34-41, 2000). They can be categorized regarding their substrate specificity, namely enzymes specific for tyrosine and/or serine/threonine residues. Many protein kinases specific for tyrosine residues are membrane associated or membrane spanning enzymes, like the epidermal growth factor receptor (EGFR/HER1). In contrast, many serine/threonine specific protein kinases are intracellular kinases involved in signal transduction processes within the cell. Regarding cancer therapy, Gilvec (Imatinib®) for treating chronic myelogenous leukemia (CML) inhibiting the bcr-abl kinase fusion protein and Iressa (Gefitinib®) inhibiting the EGFR kinase for treatment of late stage non-small-cell lung cancer (NSCLC) patients have been approved recently. Cancer is a complex disease arising after a selection process for cells with acquired functional capabilities like enhanced survival/resistance towards apoptosis and limitless proliferative potential. Thus, it is preferred to develop drugs for cancer therapy targeting several features of an established tumor. Kinase inhibitors with a defined, pathophysiological inhibition profile are preferred. As an example, Glivec potently inhibits not only the bcr-abl kinase, but also the platelet-derived growth factor receptor (PDGF-R) kinase and the c-kit receptor kinase. Other examples for kinases causally involved in tumor biology and cancer as a disease are the various vascular endothelial growth factor (VEGF) receptors, the insulin-like growth factor receptor (IGF1R) and downstream signalling kinases like phosphatidylinositol kinase (PI3K) and phosphoinositide-dependent kinase 1 (PDK1), the EGFR family including HER2, HER3 and HER4, the family of mitotic kinases including polo-like kinases (PLK) and Aurora kinase isoenzymes and raf isoenzymes including B-raf.
Within the class of serine-threonine specific signalling kinases, Akt (protein kinase B; PKB) with the isoenzmyes Akt1 (PKBα), Akt2 (PKB β) and Akt3 (PKB γ) is of high interest for therapeutic intervention. One pathway that has been shown to mediate important survival signals for mammalian cells comprises receptor tyrosine kinases like platelet-derived growth factor receptor (PDGF-R; Franke et al., Cell. 1995 Jun. 2; 81(5):727-36) or the insulin-like growth factor 1 receptor (IGF-1R; Kulik et al., Mol Cell Biol. 1997 March; 17(3):1595-606). After activation by ligand, these receptors activate the PI3K pathway. One aspect of PI3K activation is to protect cells from programmed cell death (“apoptosis”) and is hence considered to transduce a survival signal to a cell. This signal is counteracted by the lipid phosphatase PTEN (phosphatase and tensin homolog), cleaving the PI3K product PI(3, 4, 5)P3 by removing the 3′ phosphate. PTEN is deleted or functionally inactivated in many cancer cells, leading to a constitutive survival signal. The phosphatidylinositide lipid produced by PI3K can stimulate multiple kinases, including protein kinase B/Akt, which is a central downstream mediator of anti-apoptotic signals transmitted by PI3K (Datta et al., Genes Dev. 1999 Nov. 15; 13(22):2905-27. Review). The amino terminus of the Akt enzymes contains a PH domain that recruits the protein to the cell membrane by binding to the PI3K product PI(3, 4, 5)P3 and PI(3, 4,)P2 leading to a conformational change. This conformational change allows Akt to be phosphorylated at threonine 308 and serine 473 (numbering of residues according to human Akt1) by the kinase PDK1 and a postulated kinase PDK2. A number of potential substrates within the apoptotic machinery have been identified that are phosphorylated by Akt within the consensus sequence RXRXXS/T. The first Akt substrate identified was glycogen synthase kinase 3 (GSK3; Cross et al., Nature. 1995 Dec. 21-28; 378(6559):785-9) and phosphorylation of GSK3 at serine 9 results in its inactivation. Besides regulating glycogen synthesis, GSK3 is involved in the regulation of several intracellular signaling pathways including adaptor protein complex 1 (AP1), cAMP-response-element-binding protein (CREB), and the tumor suppressor gene product anaphase promoting complex (APC). Phosphorylation of the pro-apoptotic protein Bcl2 antagonist of cell death (Bad) at serine 136 by Akt creates a binding site for 14-3-3 proteins and thereby prevents Bad from binding and inhibiting the anti-apoptotic protein Bcl-xL (Datta et al., Cell. 1997 Oct. 17; 91(2):231-41). Similarly, phosphorylation of the forkhead transcription factor Forkhead in rhabdomyosarcoma-like 1 (FKHRL1) by Akt at threonine 32 and serine 253 creates a binding motif for 14-3-3 proteins resulting in cytoplasmic retention of FKHRL1 (Brunet et al., Cell. 1999 Mar. 19; 96(6):857-68); resulting in downregulation of the pro-apoptotic Fas ligand. Caspases are intracellular proteases that function as initiators and effectors of apoptosis. Akt directly phosphorylates caspase-9 at serine 196 and inhibits its protease activity (Cardone et al., Science. 1998 Nov. 13; 282(5392):1318-21). Activation of the nuclear factor kappa B (NFκB) pathway by Akt has been demonstrated by the direct association of Akt with inhibitor of nuclear factor kappa B kinase (Ikk). In vitro phosphorylation of Ikk by Akt is supposed to enhance the degradation of IκB and consequently the translocation of NFκB into the nucleus (Ozes et al., Nature. 1999 Sep. 2; 401(6748):82-5).
Constitutive activation of Akt is frequently found in human ovarian, breast (Stal et al., Breast Cancer Res., 2003, 5, R37-44), and prostate carcinomas (Malik et al., Clinc. Cancer Res., 2002, 8, 1168-1171; Thakkar et al., J. Biol. Chem., 2001, 276, 38361-38369). It is due to a complete loss of the lipid phosphatase PTEN, a negative regulator of the PI3 kinase pathway (Nesterov et al., J. Biol. Chem., 2001, 276, 10767-10774). Inhibitors of Akt are therefore promising drugs for cancer therapy as effective sensitizers or inducers of apoptosis (Beresford et al., J. Interferon Cytokine Res., 2001, 21, 313-322).
By acting as a modulator of anti-apoptotic signalling in tumor cells, the protein kinase B (PKB)/Akt is a target for cancer therapy. Compounds interfering selectively with Akt isoenzmyes have been described recently (Barneft et al. Biochem. J 2005, 385:399-408). These Akt inhibitors, particular inhibitors of Akt 1 and Akt 2, selectively sensitized tumor cells to apoptotic stimuli like Trail, Camptothecin and Doxorubicin (DeFeo-Jones et al. Mol Cancer Therap 2005, 4:271-279). Akt inhibitors therefore are expected to shift the apoptotic threshold, resensitizing tumor cells towards apoptotic stimuli of eg chemotherapeutic agents or agonists of death receptor pathways, eg Trail or agonistic DR4/5 antibodies. Nevertheless, dependent on the genetic background/molecular apperations of the tumor, Akt inhibitors might induce apoptotic cell death in monotherapy as well.